Background: The incidence of fungal infections caused by the yeasts and yeast-like species increased dramatically in immunocompromised patients, during the past several decades. However, a few of yeast and yeast-like species may colonize in skin and mucous membranes of healthy individuals. Objectives: The current study aimed at accurately identifying yeast and yeast-like species from clinical samples by molecular methods. Methods: A total of 1200 clinical samples were collected from patients with suspected fungal infection and 110 (9.16 %) yeast and yeast-like strains isolated and identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR amplification of hwp1 gene, and sequencing. Results: In total, Candida albicans (n = 46) was the most frequently isolated species followed by C. parapsilosis complex (n = 17), C. tropicalis (n = 13), C. guilliermondii (n = 12), C. glabrata (n = 4), C. krusei and C. famata each (n = 3), C. kefyr, C. haemulonii and Cutaneotrichosporon jirovecii each (n = 2), and C. stellatoidea, C. intermedia, C. sorbosivorans, C. africana, Pichia kudriavzevii, and Trichosporon asahii each (n = 1). Interestingly, C. haemulonii, a multidrug resistant fungus was isolated from cutaneous and sputum samples for the first time in Iran. Conclusions: Nowadays, with growing population at risk for fungal infections and the emergence of some less virulent or nonpathogenic and uncommon yeasts not readily distinguishable with phenotypic assays, the accurate identification using molecular methods are warranted.